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Objective:To investigate the effects of B7-H3 molecule on clear cell renal cell carcinoma (786-O) metastasis.Methods:Lentiviral transfection method was used to construct 786-O cells stably expressing low level of B7-H3 (shB7-H3 group) and a negative control cell line (shNC group). RT-qPCR, flow cytometry and Western blot were used to assess the efficiency of lentiviral transfection. CCK-8 method was used to detect the proliferation of 786-O cells in the two groups. Flow cytometry was performed to detect the changes in cell cycle. Cell scratch test and Transwell assay were used to detect the differences in cell migration and invasion. Western blot was used to detect the expression of marker proteins in the process of epithelial-mesenchymal transition (epithelial-mesenchymal transition, EMT). Changes in the expression of chemokines and their receptors were analyzed by flow cytometry and RT-qPCR. Effects of anti-CCL4 antibody on cell migration and invasion were analyzed by Transwell assay.Results:Flow cytometry showed that 786-O cells highly expressed B7-H3 molecules and the lentiviral transfection method successfully constructed the cell line with lower expression of B7-H3 (786-O-shB7-H3) and control cell line (786-O-shNC). B7-H3 molecule had no significant effect on the proliferation of 786-O cells. No significant difference in cell cycle was found between the two groups. Compared with 786-O-shNC cells, the migration and invasion ability of 786-O-shB7-H3 cells was suppressed. Moreover, the expression of EMT-related marker proteins (fibronectin and N-cadherin) was reduced and the expression of E-cadherin was increased in 786-O-shB7-H3 cells. The expression of CCL4 and its receptor CCR5 in the shB7-H3 group was lower than that in the shNC group. After intervention with anti-CCL4 antibody, the migration and invasion ability of 786-O-shNC cells was reduced, while that of 786-O-shB7-H3 cells had no significant change.Conclusions:Knocking down the expression of B7-H3 molecule had no significant effect on the proliferation of 786-O cells, but could affect the EMT process of 786-O cells and reduce tumor migration and invasion ability, thereby inhibiting tumor progression.
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@# Objective: To investigate the effect of long non-coding RNA (lncRNA) lung cancer associated transcript 1 (LUCAT1) on proliferation and migration of clear cell renal cell carcinoma (ccRCC) 786-O cells and the underlying mechanism. Methods: A total of 40 pairs of pathologically confirmed tumor tissues and corresponding adjacent normal tissues from ccRCC patients, who underwent surgical resection in the Department of Urology, the First People's Hospital of Yichang during June 2013 and June 2017, were selected for this study. ccRCC cell lines (786-O, ACHN, UM-RC-2) and normal renal epithelial KiMA cells were also used in this study. qPCR was used to detect the mRNA expressions of LUCAT1, miR-199a-5p and hypoxia inducible fator 1α (HIF-1α) in above mentioned tissues and cell lines; CCK-8 assay was used to evaluate the proliferation of 786-O cells; Transwell assay was used to evaluate the migration of 786-O cells; Dual luciferase reporter gene assay was performed to validate the relationship between LUCAT1 and miR-199a-5p; and Western blotting was conducted to detect the effect of LUCAT1 and miR-199a-5p on the protein expression of HIF-1α. Results: LUCAT1 was significantly up-regulated in ccRCC tissues and cell lines (all P<0.01), and its knockdown significantly inhibited the proliferation and migration of 786-O cells (all P<0.01). miR-199a-5p was low-expressed in ccRCC tissues and cell lines (all P<0.01), StarBase analysis showed that LUCAT1 contained a conserved target site for miR-199a-5p. miR-199a-5p exerted significant suppression on the luciferase activity of LUCAT1-Wt (P<0.01), and LUCAT1 knockdown significantly reduced miR-199a-5p expression (P< 0.01). LUCAT1 was low-expressed in 786-O cells transfected with miR-199a-5p mimics, however, it was attenuated after co-transfection with LUCAT1. The mRNA and protein expressions of HIF-1α in 786-O cells transfected with miR-199a-5p mimics were up-regulated, which was then reversed by LUCAT1 over-expression (P<0.05 or P<0.01). miR-199a-5p over-expression suppressed the proliferation and migration of 786-O cells, which was partially attenuated by LUCAT1 transfection (P<0.05 or P<0.01). Conclusion: LUCAT1 exerts oncogenic function in ccRCC via regulating miR-199a-5p/HIF-1α axis.·
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@# Objective: To explore the mechanism of miR-19a-3p regulating cell adhesion molecule 2 (CADM2) to inhibit the proliferation and metastasis of renal carcinoma cells via the AKT signaling pathway. Methods: A total of 42 patients with renal cancer admitted to Department of Nephrology, the First Affiliated Hospital of Suzhou University from April 2012 to November 2017 were enrolled to collect samples of surgically resected renal carcinoma tissues and paracancerous tissues. Expression of miR-19a-3p was detected in renal carcinoma tissues and 4 types of renal carcinoma cell lines such as 786-O by quantitative Real-time polymerase chain reaction (qPCR). The effects of miR-19a-3p knockdown on proliferation, invasion and epithelial mesenchymal transition (EMT) of renal carcinoma 786-O cells were evaluated by CCK-8 assay, Transwell assay and immunofluorescence, respectively. Subsequently, dual luciferase reporter assay was used to verify whether CADM2 was a target gene of miR-19a-3p. Furthermore, Wb was applied to detect the regulatory effect of miR-19a-3p onAKT signaling pathway through CADM2. Results: miR-19a-3p expression was significantly up-regulated in renal carcinoma tissues and cell lines (all P<0.01). Knockdown of miR-19a-3p could inhibit proliferation, invasion and EMT process of 786-O cells; furthermore, the results indicated that CADM2 was a direct target of miR-19a-3p and its expression was down-regulated (P <0.05 or P<0.01). Additionally, knockdown of miR-19a-3p obviously suppressed proliferation, migration and EMT process of 786-O cells via up-regulating CADM2 and blocking AKT pathway (all P<0.05 or P<0.01), thus alleviating the occurrence and development of renal carcinoma. Conclusion: The study demonstrates that miR-19a-3p has a high expression level in renal carcinoma tissues; knockdown of miR-19a-3p could significantly inhibit the proliferation, migration and EMT process of renal carcinoma tissues, and its mechanism may be associated with miR-19a-3p/CADM2/AKT axis.
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Objective: To investigate the anticancer effect of isoliquiritigenin (ISL) on human clear cell renal cell carcinoma 786-O cells, and explore its possible molecular mechanism. Method: Thiazolyl blue tetrazolium bromide (MTT) assay was used to detect effect of ISL (0, 10, 25,50, 75, 100 μmol·L-1) on proliferation of 786-O cells. The effect of ISL on migration and invasion of 786-O cells was detected by cell scratch test and Transwell assay. The autophagy was observed under the fluorescence microscope through acridine orange staining and Ad-GFP-LC3 transfection experiment. Western blot was used to detect the expression of autophagy related protein and analyze the changes of phosphatidylinositol-3-kinase (PI3K)/protein kinase B(Akt)/mammalian target of rapamycin (mTOR) signaling pathway to explore the possible mechanism. Result: MTT results showed that ISL could significantly inhibit the proliferation of 786-O cells in a time-dose dependent manner (PPPPPPPPConclusion: ISL can inhibit the proliferation, migration and invasion of clear cell renal carcinoma 786-O cells, and induce autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway.
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Objective To investigate the expression of miR-21 in renal clear cell carcinoma and its clinical significance as well as how miR-21 regulates the proliferation and apoptosis of 786-O renal clear cell carcinoma cell line through regulating programmed cell death 4 (PDCD4).Methods By analyzing the data of renal clear cells cancer in The Cancer Genome Atlas (TCGA)database,we compared the expression of miR-21 in renal cancer tissues and adjacent normal tissues and explored the differences in miR-21 level in renal cancer at different clinicopathological stage,T stage,N stage and M stage.We also analyzed the association between miR-21 level and survival of patients by Kaplan-Meier method and Log-rank test.786-O cells were transfected with AS-miR-21 to deplete miR-21. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis, respectively.We then measured the mRNA and protein levels of PDCD4 in 786-O cells depleted for miR-21 by qRT-PCR and Western blot,respectively,and performed a dual-luciferase assay to detect the direct regulation of PDCD4 by miR-21.Results Expression of miR-21 was significantly higher in renal cancer tissues than in adjacent tissues(P <0.0001).The expression levels of miR-21 at stage Ⅲ and stage Ⅳ renal cancer were significantly higher than that at stage Ⅰ (both P <0.0001).Moreover,miR-21 expression was positively correlated with clinicopathological stages of renal cancer by correlation analysis (r =0.262,P <0.0001 ).The correlation test indicated that miR-21 level was also positively correlated with T stage of renal cancer (r =0.250,P <0.0001 ),lymph node metastasis (N1)and distant metastasis (all P <0.0002).Patients with high miR-21 expression had significantly shorter median survival time than those with low miR-21 expression (Log-rank P < 0.001 ).Compared with control cells,786-O cells depleted for miR-21 showed significantly decreased cell proliferation (P <0.05 )and increased cell apoptosis rate (P =0.005 ).PDCD4 mRNA (P = 0.002 )and protein levels were significantly elevated in 786-O cells with down-regulated miR-21 levels.In addition,the dual-luciferase reporter assay showed that the relative luciferase intensity of PDCD4 reporter in cells transfected with AS-miR-21 was significantly higher than that of control cells (P =0.003).Conclusion miR-21 expression was up-regulated in renal cancer and correlated with clinicopathological stage and survival of patients.miR-21 promoted 786-O cell proliferation and inhibited apoptosis probably through regulating PDCD4 expression. These results indicate that miR-21 plays an important role in formation and development of renal cancer.
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Objective To investigate the expression of miR-21 in renal clear cell carcinoma and its clinical significance as well as how miR-21 regulates the proliferation and apoptosis of 786-O renal clear cell carcinoma cell line through regulating programmed cell death 4 (PDCD4).Methods By analyzing the data of renal clear cells cancer in The Cancer Genome Atlas (TCGA)database,we compared the expression of miR-21 in renal cancer tissues and adjacent normal tissues and explored the differences in miR-21 level in renal cancer at different clinicopathological stage,T stage,N stage and M stage.We also analyzed the association between miR-21 level and survival of patients by Kaplan-Meier method and Log-rank test.786-O cells were transfected with AS-miR-21 to deplete miR-21. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis, respectively.We then measured the mRNA and protein levels of PDCD4 in 786-O cells depleted for miR-21 by qRT-PCR and Western blot,respectively,and performed a dual-luciferase assay to detect the direct regulation of PDCD4 by miR-21.Results Expression of miR-21 was significantly higher in renal cancer tissues than in adjacent tissues(P <0.0001).The expression levels of miR-21 at stage Ⅲ and stage Ⅳ renal cancer were significantly higher than that at stage Ⅰ (both P <0.0001).Moreover,miR-21 expression was positively correlated with clinicopathological stages of renal cancer by correlation analysis (r =0.262,P <0.0001 ).The correlation test indicated that miR-21 level was also positively correlated with T stage of renal cancer (r =0.250,P <0.0001 ),lymph node metastasis (N1)and distant metastasis (all P <0.0002).Patients with high miR-21 expression had significantly shorter median survival time than those with low miR-21 expression (Log-rank P < 0.001 ).Compared with control cells,786-O cells depleted for miR-21 showed significantly decreased cell proliferation (P <0.05 )and increased cell apoptosis rate (P =0.005 ).PDCD4 mRNA (P = 0.002 )and protein levels were significantly elevated in 786-O cells with down-regulated miR-21 levels.In addition,the dual-luciferase reporter assay showed that the relative luciferase intensity of PDCD4 reporter in cells transfected with AS-miR-21 was significantly higher than that of control cells (P =0.003).Conclusion miR-21 expression was up-regulated in renal cancer and correlated with clinicopathological stage and survival of patients.miR-21 promoted 786-O cell proliferation and inhibited apoptosis probably through regulating PDCD4 expression. These results indicate that miR-21 plays an important role in formation and development of renal cancer.
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Objective:This study aims to determine the suitable cell line to be used in isolating cancer stem cells by comparing the characteristics of tumor stem cells in renal cell carcinoma cell lines SN12C and 786-O. Methods:The rate of sphere formation in SN12C and 786-O cells was determined in serum-free medium (SFM). The expression levels of CD133, CD44, Nanog, and Oct3/4 were investi-gated through flow cytometry. Moreover, the tumorigenicity of spheroid cell that originated from SN12C and 786-O cells was investi-gated in vivo by using a tumor model. Results:The average time of sphere formation in SFM was shorter in SN12C than in 786-O (5 days vs. 7 days). Moreover, the expression levels of CD133, CD44, Nanog, and Oct3/4 in SN12C and 786-O significantly differed (P<0.05). When transplanted in nude mice, 786-O spheres were less tumorigenic than SN12C spheres. Conclusion:SN12C spheres possess the main defining characteristics of renal cancer stem cell;thus, SN12C is the more suitable cell line to be used to isolate cancer stem cells compared with 786-O.
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Objective To investigate DNA double-strand breaks and radiosensitization in renal carcinoma 786-O cells induced by fludarabine (FA) combined with different ionizing radiations.Methods The 786-O cells were exposed to FA combined with X-ray or heavy ion beam irradiation.Flow cytometry was used to evaluate the percentage of γH2AX-positive cells and cell cycle.The neutral comet assay was used to detect DNA double-strand breaks.The colony-forming assay was used to evaluate the effects of different treatments on cell survival.Comparison between groups was made by one-way analysis of variance or Dunnet' s t test.Results Compared with FA alone or irradiation alone,FA combined with different ionizing radiations increased DNA double-strand breaks as shown by significantly increased levels of γH2AX (P=0.007,0.001);FA combined with heavy ion beam irradiation lead to a cell cycle block at the radiosensitive G2/M phase and significantly increased the expression of γH2AX in the G2/M phase (P=0.000,0.000);the neutral comet assay revealed that FA combined with irradiation significantly increased DNA sublethal damage (P=0.020,0.060);FA significantly reduced the colony-forming rate after irradiation (P=0.000,0.030;0.001,0.040).Conclusions FA enhances the effects induced by X-ray and heavy ion beam irradiation with different properties.Particularly,FA substantially enhances the cell death induced by heavy ion beam irradiation.
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Objective:To detect the effect of EphrinA1-Fc on the phosphorylation of EphA2 and extracellular signal-regulated ki-nase (ERK) in 786-O renal carcinoma cells (RCCs). Methods:The soluble ligand EphrinA1-Fc was used to inhibit the 786-O RCCs in vitro. Western blot analysis was used to examine the phosphorylation of EphA2 and ERK1/2 in the 786-O RCCs at different time points. Results:After the intervention with EphrinA1-Fc for 5, 10, 30, and 60 min, the expression of p-EphA2 increased (F=9.392, P=0.025) as well as that of p-ERK (F=4.428, P=0.041). No p-EphA2 and p-ERK expression was observed in the pre-intervention group. Conclusion:One of the possible mechanisms of the inhibitory effect of EphrinA1-Fc on tumor metastasis and recurrence involves the phosphorylation of EphA2 by EphrinA1-Fc, leading to the degradation of EphA2.